QIAGEN Multiplex PCR Kit

For highly specific and sensitive multiplex PCR without optimization requirements

No optimization required

High specificity and sensitivity with a built-in hot start

Highly suited for many types of multiplex PCR applications

Easy to use and cost-effective

The QIAGEN Multiplex PCR Kit is available in a convenient ready-to-use master mix format. The QIAGEN Multiplex PCR Master Mix includes HotStarTaq DNA Polymerase and a unique PCR buffer containing the novel synthetic Factor MP. Together with optimized salt concentrations, this additive stabilizes specifically bound primers and enables efficient extension of all primers in the reaction without the need for optimization. Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC-rich) templates, is also supplied.

The QIAGEN Multiplex PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Successful 16-plex PCR.

Multiplex PCR of 16 targets (99–955 bp) was carried out for 35 cycles using standard conditions for the QIAGEN Multiplex PCR Kit, without further optimization or using a variety of conditions with a hot-start DNA polymerase from Supplier AII. [A] Comparison using 2.5 units per 50 µl reaction of the hot-start DNA polymerase from Supplier AII and with the indicated Mg2+ concentrations. [B] Comparison using the optimized Mg2+ concentration (3.5 mM) for the hot-start DNA polymerase from Supplier AII (part A) and the indicated amounts of enzyme per 50 µl reaction. Successful results were ensured with the QIAGEN Multiplex PCR Kit. M: markers.

Genotyping transgenic mice.

Transgenic mice were screened using the QIAGEN Multiplex PCR Kit and sets of 3 primers to distinguish wild-type (wt), heterozygous mutant (ht), and homozygous mutant (hm) mice. [A] Using a primer set for the recombination activating gene 2 locus. [B] Using a primer set for the interferon-γ gene locus. M: markers. (Data kindly provided by S. zur Lage and S. Weiss, National Research Center for Biotechnology, Braunschweig, Germany.)

The QIAGEN Multiplex PCR Kit outperformed kits tested from other suppliers and ensures highly specific and sensitive multiplex PCR amplification (see figure "Successful 16-plex PCR "). The kit can be successfully used for various multiplex applications such as typing of transgenic organisms (see figure "Genotyping transgenic mice") and microsatellite analysis (see figure figure "Successful microsatellite analysis "). The master mix includes HotStarTaq DNA Polymerase for efficient amplification of multiple targets in parallel. Amplfication effiency is further improved by an innovative PCR buffer, also included in the master mix. The unique buffer ensures PCR specificity over a wide range of PCR conditions, minimizing the need for optimization. Suboptimal PCR can be improved with Q-Solution — an additive for the amplification of GC-rich templates — also provided with the kit .

The QIAGEN Multiplex PCR Kit is the first kit specifically developed for multiplex PCR and is provided in an easy-to-use master-mix format. QIAGEN Multiplex PCR Master Mix contains preoptimized concentrations of HotStarTaq DNA Polymerase and MgCl2, plus dNTPs and an innovative PCR buffer specially developed for multiplex PCR. The kit enables success in multiplex PCR at the first attempt. There is no need to optimize reaction conditions (e.g., the concentrations of primers, Mg2+, and Taq DNA polymerase) and cycling parameters due to unique preoptimized reagents included in the kit.

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase is a modified form of Taq DNA polymerase and has no polymerase activity at ambient temperatures. This prevents extension of nonspecifically annealed primers and primer dimers formed at low temperatures during PCR setup and the initial PCR cycle. HotStarTaq DNA Polymerase is activated by a 15-minute incubation at 95°C which can be incorporated into any existing thermal-cycler program.

Multiplex PCR Buffer

This special buffer contains an optimized combination of K+ and NH4+, as well as the unique PCR additive, Factor MP, which increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq DNA Polymerase (see figure "Stable and efficient primer annealing"). The innovative buffer maintains specific amplification in every cycle of PCR by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg2+ concentration is therefore often minimal or not required.

Q-Solution

Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA, is also provided with HotStarTaq DNA Polymerase. This unique reagent improves suboptimal PCR caused by templates that have a high degree of secondary structure or GC-rich templates. Unlike other commonly used PCR additives such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed.

Procedure

The QIAGEN Multiplex PCR Kit is provided in a ready-to-use, preoptimized master mix for greater convenience. Use of a master mix saves time, simplifies handling for reaction setup, and increases reproducibility by eliminating many possible sources of pipetting errors and contamination — pipetting steps are minimized and tedious calculations are eliminated. Only primers and template need to be added to prepare the final amplification mix. The master mix can be stored at 2–8°C, allowing even faster setup of multiplex PCR assays. The streamlined, step-by-step protocol provided with the kit ensures fast and easy PCR setup. Reactions can be set up at room temperature, ensuring greater convenience and ease of use. HotStarTaq DNA Polymerase is activated by a 15-minute, 95°C incubation step, which can easily be incorporated into existing thermal cycling programs.

Applications

The QIAGEN Multiplex PCR Kit is highly suited for various multiplex applications, including: